Isolation of intact m-RNA is required for many biological procedures. For example, m-RNA can be used for cDNA synthesis, in vitro translation, and/or "Northern" hybridization. See e.g. J. Sambrook et al., Molecular Cloning: A Labratory Manual (Cold Spring Harbor 1989). The disclosures of this publication and the disclosures of all other publications recited herein are incorporated by reference as if fully set forth herein.
Messenger RNA is a particularly difficult macromolecule to isolate from cells. This is due to the presence of RNase in cells which will rapidly degrade the RNA upon disruption of the cellular compartments. Therefore, cells in which RNA is to be isolated have been disrupted in the presence of a number of different denaturants (i.e. phenol, LiCl, SDS) and RNase inhibitors (i.e. heparin, iodoacetate, diethyl pyrocarbonate, polyvinyl sulfate, aurin tricarboxylic acid).
The best denaturant/inhibitors known are guanidine salts (e.g. guanidine isothiocyanate a/k/a guanidinium thiocyanate). They are used to isolate an intermediate product, total RNA. See P. Chomczynski et al., 162 Anal Biochem. 156-159 (1987); J. Chirgwin et al., 18 Biochemistry 294-5299 (1979). After cell lysis using the guanidine salt, cellular matter is subjected to extended equilibrium centrifugation in CsCl or CsTFA during which total RNA becomes pelleted at the bottom of the tube.
Following aspiration of the supernatant, the total RNA pellet is redissolved in a buffer. This separates total RNA from guanidine salts (and unfortunately thus removes RNase inhibitors). Once total cellular RNA has been isolated, the mRNA can then be purified by the passage of total RNA over a column of oligo d(T)-cellulose. H. Aviv, et al., 69 P.N.A.S. USA 1408-1412 (1972). This method capitalizes on the presence of long stretches of adenyl residues at the 3' end eukaryotic m-RNA. Under suitable conditions of high salt, hydrogen bonding between the adenylated mRNA and the oligo d(T) will form, and the mRNA will be retained on the column. The mRNA is subsequently eluted in the presence of a no-salt buffer.
The above method of mRNA isolation has been improved by purification of mRNA from total RNA on a column (which is oligo d(T) bound to a solid support) in spun column format. Such columns are available from Pharmacia, or from Clonetech, or 5'-3'. However, this method of total RNA isolation followed by mRNA purification can still take many hours (or sometimes days) to complete.
Attempts have been made to reduce the time needed for m-RNA isolation. See K. Jakobsen et al., 18 Nuc. Acids Res. 3669 (Jun. 25, 1990); J. Badley, 6 BioTechniques 114-116 (1988). However, these approaches did not involve guanidine moieties. Instead they relied upon proteases or LiCl to disrupt the cell, neither of which gave adequate RNase protection. Further, the protease system had other problems (e.g. inadequate lysing capability).
Thus, a need still exists for an improved mRNA isolation technique.